Diagnostic and therapeutic use of ma onconeuronal antigents for neurodegenerative diseases

ABSTRACT

The present invention discloses the differential expression of the onconeuronal antigen Ma2 gene in specific brain regions of Alzheimer&#39;s disease patients. Based on this finding, this invention provides a method for diagnosing or prognosticating a neurodegenerative disease, in particular Alzheimer&#39;s disease, in a subject, or for determining whether a subject is at increased risk of developing such a disease. Furthermore, this invention provides therapeutic and prophylactic methods for treating or preventing Alzheimer&#39;s disease and related neurodegenerative disorders using a gene coding for an onconeuronal antigen, in particular Ma2 onconeuronal antigen. A method of screening for modulating agents of neurodegenerative diseases is also disclosed.

The present invention relates to methods of diagnosing, prognosticatingand monitoring the progression of neurodegenerative diseases in asubject. Furthermore, methods of therapy control and screening formodulating agents of neurodegenerative diseases are provided. Theinvention also discloses pharmaceutical compositions, kits, andrecombinant animal models.

Neurodegenerative diseases, in particular Alzheimer's disease (AD), havea strongly debilitating impact on a patient's life. Furthermore, thesediseases constitute an enormous health, social, and economic burden. ADis the most common neurodegenerative disease, accounting for about 70%of all dementia cases, and it is probably the most devastatingage-related neurodegenerative condition affecting about 10% of thepopulation over 65 years of age and up to 45% over age 85 (for a recentreview see Vickers et al., Progress in Neurobiology 2000, 60: 139-165).Presently, this amounts to an estimated 12 million cases in the US,Europe, and Japan. This situation will inevitably worsen with thedemographic increase in the number of old people (“aging of the babyboomers”) in developed countries. The neuropathological hallmarks thatoccur in the brains of individuals with AD are senile plaques, composedof amyloid-P protein, and profound cytoskeletal changes coinciding withthe appearance of abnormal filamentous structures and the formation ofneurofibrillary tangles.

The amyloid-β (Aβ) protein evolves from the cleavage of the amyloidprecursor protein (APP) by different kinds of proteases. The cleavage bythe β/γ-secretase leads to the formation of Aβ peptides of differentlengths, typically a short more soluble and slow aggregating peptideconsisting of 40 amino acids and a longer 42 amino acid peptide, whichrapidly aggregates outside the cells, forming the characteristic amyloidplaques (Selkoe, Physiological Rev 2001, 81: 741-66; Greenfield et al.,Frontiers Bioscience 2000, 5: D72-83). Two types of plaques, diffuseplaques and neuritic plaques, can be detected in the brain of ADpatients, the latter ones being the classical, most prevalent type. Theyare primarily found in the cerebral cortex and hippocampus. The neuriticplaques have a diameter of 50 μm to 200 μm and are composed of insolublefibrillar amyloids, fragments of dead neurons, microglia and astrocytes,and other components such as neurotransmitters, apolipoprotein E,glycosaminoglycans, α1-antichymotrypsin and others. The generation oftoxic Aβ deposits in the brain starts very early in the course of AD,and it is discussed to be a key player for the subsequent destructiveprocesses leading to AD pathology. The other pathological hallmarks ofAD are neurofibrillary tangles (NFTs) and abnormal neurites, describedas neuropil threads (Braak and Braak, Acta Neuropathol 1991, 82:239-259). NFTs emerge inside neurons and consist of chemically alteredtau, which forms paired helical filaments twisted around each other.Along the formation of NFTs, a loss of neurons can be observed. It isdiscussed that said neuron loss may be due to a damagedmicrotubule-associated transport system (Johnson and Jenkins, JAlzheimers Dis 1996, 1: 38-58; Johnson and Hartigan, J Alzheimers Dis1999, 1: 329-351). The appearance of neurofibrillary tangles and theirincreasing number correlates well with the clinical severity of AD(Schmitt et al., Neurology 2000, 55: 370-376).

AD is a progressive disease that is associated with early deficits inmemory formation and ultimately leads to the complete erosion of highercognitive function. The cognitive disturbances include among otherthings memory impairment, aphasia, agnosia and the loss of executivefunctioning. A characteristic feature of the pathogenesis of AD is theselective vulnerability of particular brain regions and subpopulationsof nerve cells to the degenerative process. Specifically, the temporallobe region and the hippocampus are affected early and more severelyduring the progression of the disease. On the other hand, neurons withinthe frontal cortex, occipital cortex, and the cerebellum remain largelyintact and are protected from neurodegeneration (Terry et al., Annals ofNeurology 1981, 10: 184-92).

The age of onset of AD may vary within a range of 50 years, withearly-onset AD occurring in people younger than 65 years of age, andlate-onset of AD occurring in those older than 65 years. About 10% ofall AD cases suffer from early-onset AD, with only 1-2% being familial,inherited cases.

Currently, there is no cure for AD, nor is there an effective treatmentto halt the progression of AD or even to diagnose AD ante-mortem withhigh probability. Several risk factors have been identified thatpredispose an individual to develop AD, among them most prominently theepsilon 4 allele of the three different existing alleles (epsilon 2, 3,and 4) of the apolipoprotein E gene (ApoE) (Strittmatter et al., ProcNatl Acad Sci USA 1993, 90: 1977-81; Roses, Ann NY Acad Sci 1998, 855:738-43). The polymorphic plasmaprotein ApoE plays a role in theintercellular cholesterol and phospholipid transport by bindinglow-density lipoprotein receptors, and it seems to play a role inneurite growth and regeneration. Efforts to detect furthersusceptibility genes and disease-linked polymorphisms, lead to theassumption that specific regions and genes on human chromosomes 10 and12 may be associated with late-onset AD (Myers et al., Science 2000,290: 2304-5; Bertram et al., Science 2000, 290: 2303; Scott et al., Am JHum Genet 2000, 66: 922-32).

Although there are rare examples of early-onset AD which have beenattributed to genetic defects in the genes for amyloid precursor protein(APP) on chromosome 21, presenilin-1 on chromosome 14, and presenilin-2on chromosome 1, the prevalent form of late-onset sporadic AD is ofhitherto unknown etiologic origin. The mutations found to date accountfor only half of the familial AD cases, which is less than 2% of all ADpatients. The late onset and complex pathogenesis of neurodegenerativedisorders pose a formidable challenge to the development of therapeuticand diagnostic agents. It is pivotal to expand the pool of potentialdrug targets and diagnostic markers. It is therefore an object of thepresent invention to provide insight into the pathogenesis ofneurological diseases and to provide methods, materials, agents,compositions, and animal models which are suited inter alia for thediagnosis and development of a treatment of these diseases. This objecthas been solved by the features of the independent claims. The subclaimsdefine preferred embodiments of the present invention.

For long, it has been known that a relationship exists between specificdegenerative processes of the nervous system and the presence of cancerin the body. These extremely rare conditions are collectively termed‘paraneoplastic neurologic disease’ (PND). It became apparent that PNDpatients harbored high-titer antibodies in their sera and cerebrospinalfluids that were directed to proteins expressed by both, the tumor cellsand the degenerating neurons. These proteins were named onconeuronalantigens (Darnell et al., J. Neurosci. 1991, 11: 1224-1230).

An emerging model for the pathogenesis of the PNDs comprises three mainaspects, namely (i) normally, onconeuronal antigens are solely expressedin neurons which represent an immuneprivileged site; therefore, theseantigens are recognized by the immune system as foreign once they areexpressed on tumor cells; (ii) the immune response to the onconeuronalantigens ectopically expressed on tumor cells provides tumor immunity;(iii) some patients with onconeuronal-antigen-based tumor immunity showa breakdown in their immunological tolerance to neurons, thus developingan humoral and/or CD8+ T-cell mediated autoimmune neurologic disease.Several different families of onconeuronal antigens have beencharacterized that are typically associated with one particularneurological condition and type of tumor (for review, Musunuru andDarnell, Annu. Rev. Neurosci 2001, 24: 239-262).

Paraneoplastic opsoclonus-myoclonus ataxia and paraneoplasticencephalomyelitis and sensory neuropathy are PNDs associated with theonconeuronal antigens Nova and Hu, respectively, and small-cell lungcancer. Nova and Hu antigens possess RNA binding motifs and thus may beinvolved in aspects of RNA localization and/or function in neurons. Theparaneoplastic cerebellar degeneration antigen Cdr2 is associated withbreast and ovarian cancer and may play a role in neuronal apoptosis.

Similarly, antibodies to the growing Ma family of onconeuronal antigensidentify a PND, i.e. encephalitis, that affects the limbic system, brainstem, and cerebellum. In particular, immunity to Ma2 is predominantlyassociated with limbic and brainstem encephalitis and germ-cell tumorsof the testis (Rosenfeld, Ann. Neurol. 2001, 50: 339-348).

Ma1-3 and MAP-1 genes (standing for Modulator of Apoptosis-1; Tan etal., J. Biol. Chem. 2001, 276: 2802-2807) code for a family ofhomologous proteins sharing potential phosphorylation sites for proteinkinase C, casein kinase II, and cAMP-dependent protein kinase and theBH-3-like (BH: Bcl-2 homology) signature motif. Their appearance in‘speckled bodies’ within cell nuclei lead to the speculation that Mafamily proteins are involved in pre-mRNA processing (Rosenfeld, Ann.Neurol. 2001, 50: 339-348). On the transcriptional level, the tissuedistribution of the Ma onconeuronal antigens in humans is as follows:Ma1, expressed in brain and testis; Ma2, expressed as a 6.5 kb mRNA inbrain; Ma3, expressed in brain, testis, trachea, kidney, and heart;MAP-1, expressed in heart, brain, skeletal muscle, kidney, and pancreas(Rosenfeld, Ann. Neurol. 2001, 50:339-348; Tan et al., J. Biol. Chem.2001, 276:2802-2807).

The nucleotide sequence of the Ma2 gene was initially determined byNagase, et al. (DNA Research 1998, 5:355-364) from the KIAA0883 clonederived from a human brain cDNA library. The authors deposited thesequence in the GenBank database with the accession number AB020690. TheMa2 gene codes for a polypeptide of 364 amino acids in length with apredicted molecular weight of 41.5 kDa (GenBank accession number:BAA74906).

The onconeuronal antigen Ma2 is of particular interest inasmuch itrepresents the dominant autoantigen among the different Ma familymembers. There is a major immunodominant region in the N-terminalportion of Ma2. All patients with autoimmunity to Ma proteins developantibodies against this particular region (Voltz et al., New Engl J Med1999, 340: 1788-1795; Rosenfeld, Ann. Neurol. 2001, 50: 339-348). Whilethe majority of PND patients with Ma2 directed autoantibodies only hadgerm-cell tumors of the testis, patients with additional Ma1 and Ma3directed autoimmunity had tumors other than germ cell neoplasms (andprominent brainstem and cerebellar symptoms rather thanlimbic-hypothalamic-brainstem symptoms). A histopathological examinationof Ma2-associated limbic dysfunction revealed mononuclear inflammatoryinfiltrates, astrogliosis, and neuronal degeneration (Voltz et al., NewEngl J Med 1999, 340: 1788-1795). In most cases the neurologic symptomspreceded the diagnosis of the tumor.

Based on the above information, Ma2 in particular can be regarded as aselective and well-documented marker protein for neuronal cells in thebrain. To date, however, no experiments have been described that show arelationship between a differential expression of the Ma2 gene or anyother Ma family member gene and the pathology of neurodegenerativediseases like Alzheimer's disease, Parkinson's disease, Huntington'sdisease, amyothrophic lateral sclerosis, Pick's disease, frontotemporaldementia, progressive nuclear palsy, cerebro-vascular dementia, orcorticobasal degeneration. All of these neurodegenerative diseases havean etiology clearly distinct from the acute inflammatory processes thatcharacterize PNDs. Likewise, no experiments have yet been described thatsuggest a relationship between a dysregulation of Ma2 gene expressionand the pathology of said neurodegenerative diseases. To date nomutations in the Ma family member genes have been found to be associatedwith a pathological phenotype of said neurodegenerative disorders.

The singular forms “a”, “an”, and “the” as used herein and in the claimsinclude plural reference unless the context dictates otherwise. Forexample, “a cell” means as well a plurality of cells, and so forth. Theterm “and/or” as used in the present specification and in the claimsimplies that the phrases before and after this term are to be consideredeither as alternatives or in combination. For instance, the wording“determination of a level and/or an activity” means that either only alevel, or only an activity, or both a level and an activity aredetermined. The term “level” as used herein is meant to comprise a gageof, or a measure of the amount of, or a concentration of a transcriptionproduct, for instance an mRNA, or a translation product, for instance aprotein or polypeptide. The term “activity” as used herein shall beunderstood as a measure for the ability of a transcription product or atranslation product to produce a biological effect or a measure for alevel of biologically active molecules. The term “activity” also refersto enzymatic activity. The terms “level” and/or “activity” as usedherein further refer to gene expression levels or gene activity. Geneexpression can be defined as the utilization of the informationcontained in a gene by transcription and translation leading to theproduction of a gene product. “Dysregulation” shall mean an upregulationor downregulation of gene expression. A gene product comprises eitherRNA or protein and is the result of expression of a gene. The amount ofa gene product can be used to measure how active a gene is. The term“gene” as used in the present specification and in the claims comprisesboth coding regions (exons) as well as non-coding regions (e.g.non-coding regulatory elements such as promoters or enhancers, introns,leader and trailer sequences). The term “ORF” is an acronym for “openreading frame” and refers to a nucleic acid sequence that does notpossess a stop codon in at least one reading frame and therefore canpotentially be translated into a sequence of amino acids. “Regulatoryelements” shall comprise inducible and non-inducible promoters,enhancers, operators, and other elements that drive and regulate geneexpression. The term “fragment” as used herein is meant to comprise e.g.an alternatively spliced, or truncated, or otherwise cleavedtranscription product or translation product. The term “derivative” asused herein refers to a mutant, or an RNA-edited, or a chemicallymodified, or otherwise altered transcription product, or to a mutant, orchemically modified, or otherwise altered translation product. Forinstance, a “derivative” may be generated by processes such as alteredphosphorylation, or glycosylation, or acetylation, or lipidation, or byaltered signal peptide cleavage or other types of maturation cleavage.These processes may occur post-translationally. The term “modulator” asused in the present invention and in the claims refers to a moleculecapable of changing or altering the level and/or the activity of a gene,or a transcription product of a gene, or a translation product of agene. Preferably, a “modulator” is capable of changing or altering thebiological activity of a transcription product or a translation productof a gene. Said modulation, for instance, may be an increase or adecrease in enzyme activity, a change in binding characteristics, or anyother change or alteration in the biological, functional, orimmunological properties of said translation product of a gene. Theterms “agent”, “reagent”, or “compound” refer to any substance,chemical, composition or extract that have a positive or negativebiological effect on a cell, tissue, body fluid, or within the contextof any biological system, or any assay system examined. They can beagonists, antagonists, partial agonists or inverse agonists of a target.Such agents, reagents, or compounds may be nucleic acids, natural orsynthetic peptides or protein complexes, or fusion proteins. They mayalso be antibodies, organic or anorganic molecules or compositions,small molecules, drugs and any combinations of any of said agents above.They may be used for testing, for diagnostic or for therapeuticpurposes. The terms “oligonucleotide primer” or “primer” refer to shortnucleic acid sequences which can anneal to a given target polynucleotideby hybridization of the complementary base pairs and can be extended bya polymerase. They may be chosen to be specific to a particular sequenceor they may be randomly selected, e.g. they will prime all possiblesequences in a mix. The length of primers used herein may vary from 10nucleotides to 80 nucleotides. “Probes” are short nucleic acid sequencesof the nucleic acid sequences described and disclosed herein orsequences complementary therewith. They may comprise full lengthsequences, or fragments, derivatives, isoforms, or variants of a givensequence. The identification of hybridization complexes between a“probe” and an assayed sample allows the detection of the presence ofother similar sequences within that sample. As used herein, “homolog orhomology” is a term used in the art to describe the relatedness of anucleotide or peptide sequence to another nucleotide or peptidesequence, which is determined by the degree of identity and/orsimilarity between said sequences compared. The term “variant” as usedherein refers to any polypeptide or protein, in reference topolypeptides and proteins disclosed in the present invention, in whichone or more amino acids are added and/or substituted and/or deletedand/or inserted at the N-terminus, and/or the C-terminus, and/or withinthe native amino acid sequences of the native polypeptides or proteinsof the present invention. Furthermore, the term “variant” shall includeany shorter or longer version of a polypeptide or protein. “Variants”shall also comprise a sequence that has at least about 80% sequenceidentity, more preferably at least about 90% sequence identity, and mostpreferably at least about 95% sequence identity with the amino acidsequences of the Ma onconeuronal antigen. “Variants” of a proteinmolecule include, for example, proteins with conservative amino acidsubstitutions in highly conservative regions. “Proteins andpolypeptides” of the present invention include variants, fragments andchemical derivatives of the protein comprising the amino acid sequencesof Ma onconeuronal antigen. They can include proteins and polypeptideswhich can be isolated from nature or be produced by recombinant and/orsynthetic means. Native proteins or polypeptides refer tonaturally-occurring truncated or secreted forms, naturally occurringvariant forms (e.g. splice-variants) and naturally occurring allelicvariants. The term “isolated” as used herein is considered to refer tomolecules that are removed from their natural environment, i.e. isolatedfrom a cell or from a living organism in which they normally occur, andthat are separated or essentially purified from the coexistingcomponents with which they are found to be associated in nature. Thisnotion further means that the sequences encoding such molecules can belinked by the hand of man to polynucleotides, to which they are notlinked in their natural state, and that such molecules can be producedby recombinant and/or synthetic means. Even if for said purposes thosesequences may be introduced into living or non-living organisms bymethods known to those skilled in the art, and even if those sequencesare still present in said organisms, they are still considered to beisolated. In the present invention, the terms “risk”, “susceptibility”,and “predisposition” are tantamount and are used with respect to theprobability of developing a neurodegenerative disease, preferablyAlzheimer's disease.

The term ‘AD’ shall mean Alzheimer's disease. “AD-type neuropathology”as used herein refers to neuropathological, neurophysiological,histopathological and clinical hallmarks as described in the instantinvention and as commonly known from state-of-the-art literature (see:Iqbal, Swaab, Winblad and Wisniewski, Alzheimer's Disease and RelatedDisorders (Etiology, Pathogenesis and Therapeutics), Wiley & Sons, NewYork, Weinheim, Toronto, 1999; Scinto and Daffner, Early Diagnosis ofAlzheimer's Disease, Humana Press, Totowa, N.J., 2000; Mayeux andChristen, Epidemiology of Alzheimer's Disease: From Gene to Prevention,Springer Press, Berlin, Heidelberg, New York, 1999; Younkin, Tanzi andChristen, Presenilins and Alzheimer's Disease, Springer Press, Berlin,Heidelberg, New York, 1998).

Neurodegenerative diseases or disorders according to the presentinvention comprise Alzheimer's disease, Parkinson's disease,Huntington's disease, amyotrophic lateral sclerosis, Pick's disease,fronto-temporal dementia, progressive nuclear palsy, corticobasaldegeneration, cerebro-vascular dementia, multiple system atrophy,argyrophilic grain dementia and other tauopathies, and mild-cognitiveimpairment. Further conditions involving neurodegenerative processesare, for instance, age-related macular degeneration, narcolepsy, motorneuron diseases, prion diseases, traumatic nerve injury and repair, andmultiple sclerosis.

In one aspect, the invention features a method of diagnosing orprognosticating a neurodegenerative disease in a subject, or determiningwhether a subject is at increased risk of developing said disease. Themethod comprises: determining a level, or an activity, or both saidlevel and said activity of (i) a transcription product of a gene codingfor a Ma onconeuronal antigen, and/or of (ii) a translation product of agene coding for a Ma onconeuronal antigen, and/or of (iii) a fragment,or derivative, or variant of said transcription or translation productin a sample from said subject and comparing said level, and/or saidactivity to a reference value representing a known disease or healthstatus, thereby diagnosing or prognosticating said neurodegenerativedisease in said subject, or determining whether said subject is atincreased risk of developing said neurodegenerative disease.

The invention also relates to the construction and the use of primersand probes which are unique to the nucleic acid sequences, or fragmentsor variants thereof, as disclosed in the present invention. Theoligonucleotide primers and/or probes can be labeled specifically withfluorescent, bioluminescent, magnetic, or radioactive substances. Theinvention further relates to the detection and the production of saidnucleic acid sequences, or fragments and variants thereof, using saidspecific oligonucleotide primers in appropriate combinations.PCR-analysis, a method well known to those skilled in the art, can beperformed with said primer combinations to amplify said gene specificnucleic acid sequences from a sample containing nucleic acids. Suchsample may be derived either from healthy or diseased subjects. Whetheran amplification results in a specific nucleic acid product or not, andwhether a fragment of different length can be obtained or not, may beindicative for a neurodegenerative disease, in particular Alzheimer'sdisease. Thus, the invention provides nucleic acid sequences,oligonucleotide primers, and probes of at least 10 bases in length up tothe entire coding and gene sequences, useful for the detection of genemutations and single nucleotide polymorphisms in a given samplecomprising nucleic acid sequences to be examined, which may beassociated with neurodegenerative diseases, in particular Alzheimersdisease. This feature has utility for developing rapid DNA-baseddiagnostic tests, preferably also in the format of a kit.

In a further aspect, the invention features a method of monitoring theprogression of a neurodegenerative disease in a subject. A level, or anactivity, or both said level and said activity, of (i) a transcriptionproduct of a gene coding for a Ma onconeuronal antigen, and/or of (ii) atranslation product of a gene coding for a Ma onconeuronal antigen,and/or of (iii) a fragment, or derivative, or variant of saidtranscription or translation product in a sample from said subject isdetermined. Said level and/or said activity is compared to a referencevalue representing a known disease or health status. Thereby theprogression of said neurodegenerative disease in said subject ismonitored.

In still a further aspect, the invention features a method of evaluatinga treatment for a neurodegenerative disease, comprising determining alevel, or an activity, or both said level and said activity of (i) atranscription product of a gene coding for a Ma onconeuronal antigen,and/or of (ii) a translation product of a gene coding for a Maonconeuronal antigen, and/or of (iii) a fragment, or derivative, orvariant of said transcription or translation product in a sampleobtained from a subject being treated for said disease. Said level, orsaid activity, or both said level and said activity are compared to areference value representing a known disease or health status, therebyevaluating the treatment for said neurodegenerative disease.

In a further preferred embodiment of the herein claimed methods, kits,recombinant animals, molecules, assays, and uses of the instantinvention, said neurodegenerative disease or disorder is Alzheimer'sdisease, and said subjects suffer from Alzheimer's disease.

In a preferred embodiment of the herein claimed methods, kits,recombinant animals, molecules, assays, and uses of the instantinvention, said gene coding for the onconeuronal antigen is a member ofthe Ma family of onconeuronal antigens, particularly Ma2.

The present invention discloses the detection and differentialexpression and regulation of the Ma2 gene in specific brain regions ofAlzheimer's disease patients. Consequently, the Ma2 gene and itscorresponding transcription and/or translation products may have acausative role in the regional selective neuronal degeneration typicallyobserved in Alzheimer's disease. Alternatively, Ma2 may confer aneuroprotective function to the remaining surviving nerve cells. Basedon these disclosures, the present invention has utility for thediagnostic evaluation and prognosis as well as for the identification ofa predisposition to a neurodegenerative disease, in particularAlzheimer's disease. Furthermore, the present invention provides methodsfor the diagnostic monitoring of patients undergoing treatment for sucha disease.

It is preferred that the sample to be analyzed and determined isselected from the group comprising brain tissue or other body cells. Thesample can also comprise cerebrospinal fluid or other body fluidsincluding saliva, urine, serum plasma, or mucus. Preferably, the methodsof diagnosis, prognosis, monitoring the progression or evaluating atreatment for a neurodegenerative disease, according to the instantinvention, can be practiced ex corpore, and such methods preferablyrelate to samples, for instance, body fluids or cells, removed,collected, or isolated from a subject or patient.

In further preferred embodiments, said reference value is that of alevel, or an activity, or both said level and said activity of (i) atranscription product of a gene coding for a Ma onconeuronal antigen,and/or of (ii) a translation product of a gene coding for a Maonconeuronal antigen, and/or of (iii) a fragment, or derivative, orvariant of said transcription or translation product in a sample from asubject not suffering from said neurodegenerative disease.

In preferred embodiments, an alteration in the level and/or activity ofa transcription product of the gene coding for a Ma onconeuronal antigenand/or a translation product of the gene coding for a Ma onconeuronalantigen in a sample cell, or tissue, or body fluid from said subjectrelative to a reference value representing a known health statusindicates a diagnosis, or prognosis, or increased risk of becomingdiseased with a neurodegenerative disease, particularly Alzheimer'sdisease.

In preferred embodiments, measurement of the level of transcriptionproducts of a gene coding for a Ma onconeuronal antigen is performed ina sample from a subject using a quantitative PCR-analysis with primercombinations to amplify said gene specific sequences from cDNA obtainedby reverse transcription of RNA extracted from a sample of a subject. ANorthern blot with probes specific for said gene can also be applied. Itmight also be preferred to measure transcription products by means ofchip-based micro-array technologies. These techniques are known to thoseof ordinary skill in the art (see Sambrook and Russell, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2001; Schena M., Microarray Biochip Technology,Eaton Publishing, Natick, Mass., 2000). An example of an immunoassay isthe detection and measurement of enzyme activity as disclosed anddescribed in the patent application WO 02/14543.

Furthermore, the level of a translation product of a gene coding for aMa onconeuronal antigen and/or a fragment, or derivative, or variant ofsaid translation product, and/or the level of activity of saidtranslation product and/or a fragment, or derivative, or variant of saidtranslation product, can be detected using an immunoassay, an activityassay and/or a binding assay. These assays can measure the amount ofbinding between said protein molecule and an anti-protein antibody bythe use of enzymatic, chromodynamic, radioactive, magnetic, orluminescent labels which are attached to either the anti-proteinantibody or a secondary antibody which binds the anti-protein antibody.In addition, other high affinity ligands may be used. Immunoassays whichcan be used include e.g. ELISAs, Western blots and other techniquesknown to those of ordinary skill in the art (see Harlow and Lane, UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1999 and Edwards R, Immunodiagnostics: APractical Approach, Oxford University Press, Oxford; England, 1999). Allthese detection techniques may also be employed in the format ofmicroarrays, protein-arrays, antibody microarrays, tissue microarrays,electronic biochip or protein-chip based technologies (see Schena M.,Microarray Biochip Technology, Eaton Publishing, Natick, Mass., 2000).

In a preferred embodiment, the level, or the activity, or both saidlevel and said activity of (i) a transcription product of a gene codingfor a Ma onconeuronal antigen, and/or of (ii) a translation product of agene coding for a Ma onconeuronal antigen, and/or of (iii) a fragment,or derivative, or variant of said transcription or translation productin a series of samples taken from said subject over a period of time iscompared, in order to monitor the progression of said disease. Infurther preferred embodiments, said subject receives a treatment priorto one or more of said sample gatherings. In yet another preferredembodiment, said level and/or activity is determined before and aftersaid treatment of said subject.

In another aspect, the invention features a kit for diagnosing orprognosticating neurodegenerative diseases, in particular Alzheimer'sdisease, in a subject, or determining the propensity or predispositionof a subject to develop a neurodegenerative disease, in particularAlzheimer's disease, said kit comprising:

-   -   (a) at least one reagent which is selected from the group        consisting of (i) reagents that selectively detect a        transcription product of a gene coding for a Ma onconeuronal        antigen, and (ii) reagents that selectively detect a translation        product of a gene coding for a Ma onconeuronal antigen; and    -   (b) instruction for diagnosing, or prognosticating a        neurodegenerative disease, in particular Alzheimer's disease, or        determining the propensity or predisposition of a subject to        develop such a disease by        -   detecting a level, or an activity, or both said level and            said activity, of said transcription product and/or said            translation product of a gene coding for a Ma onconeuronal            antigen, in a sample from said subject; and        -   diagnosing or prognosticating a neurodegenerative disease,            in particular Alzheimer's disease, or determining the            propensity or predisposition of said subject to develop such            a disease,            wherein a varied level, or activity, or both said level and            said activity, of said transcription product and/or said            translation product compared to a reference value            representing a known health status; or a level, or activity,            or both said level and said activity, of said transcription            product and/or said translation product similar or equal to            a reference value representing a known disease status,            indicates a diagnosis or prognosis of a neurodegenerative            disease, in particular Alzheimer's disease, or an increased            propensity or predisposition of developing such a disease.            The kit, according to the present invention, may be            particularly useful for the identification of individuals            that are at risk of developing a neurodegenerative disease,            in particular Alzheimer's disease. Consequently, the kit,            according to the invention, may serve as a means for            targeting identified individuals for early preventive            measures or therapeutic intervention prior to disease onset,            before irreversible damage in the course of the disease has            been inflicted. Furthermore, in preferred embodiments, the            kit featured in the invention is useful for monitoring a            progression of a neurodegenerative disease, in particular            Alzheimer's disease, in a subject, as well as monitoring            success or failure of therapeutic treatment for such a            disease of said subject.

In another aspect, the invention features a method of treating orpreventing a neurodegenerative disease, in particular Alzheimer'sdisease, in a subject comprising the administration to said subject in atherapeutically or prophylactically effective amount of an agent oragents which directly or indirectly affect a level, or an activity, orboth said level and said activity, of (i) a gene coding for a Maonconeuronal antigen, and/or (ii) a transcription product of a genecoding for a Ma onconeuronal antigen, and/or (iii) a translation productof said gene, and/or (iv) a fragment, or derivative, or variant of (i)to (iii). Said agent may comprise a small molecule, or it may alsocomprise a peptide, an oligopeptide, or a polypeptide. Said peptide,oligopeptide, or polypeptide may comprise an amino acid sequence of atranslation product of the gene coding for a Ma onconeuronal antigenprotein, or a fragment, or derivative, or a variant thereof. An agentfor treating or preventing a neurodegenerative disease, in particularAD, according to the instant invention, may also consist of anucleotide, an oligonucleotide, or a polynucleotide. Saidoligonucleotide or polynucleotide may comprise a nucleotide sequence ofthe gene coding for a Ma onconeuronal antigen protein, either in senseorientation or in antisense orientation.

In preferred embodiments, the method comprises the application of per seknown methods of gene therapy and/or antisense nucleic acid technologyto administer said agent or agents. In general, gene therapy includesseveral approaches: molecular replacement of a mutated gene, addition ofa new gene resulting in the synthesis of a therapeutic protein, andmodulation of endogenous cellular gene expression by recombinantexpression methods or by drugs. Gene-transfer techniques are describedin detail (see e.g. Behr, Acc Chem Res 1993, 26: 274-278 and Mulligan,Science 1993, 260: 926-931) and include direct gene-transfer techniquessuch as mechanical microinjection of DNA into a cell as well as indirecttechniques employing biological vectors (like recombinant viruses,especially retroviruses) or model liposomes, or techniques based ontransfection with DNA coprecipitation with polycations, cell membranepertubation by chemical (solvents, detergents, polymers, enzymes) orphysical means (mechanic, osmotic, thermic, electric shocks). Thepostnatal gene transfer into the central nervous system has beendescribed in detail (see e.g. Wolff, Curr Opin Neurobiol 1993, 3:743-748).

In particular, the invention features a method of treating or preventinga neurodegenerative disease by means of antisense nucleic acid therapy,i.e. the down-regulation of an inappropriately expressed or defectivegene by the introduction of antisense nucleic acids or derivativesthereof into certain critical cells (see e.g. Gillespie, DN&P 1992, 5:389-395; Agrawal and Akhtar, Trends Biotechnol 1995, 13: 197-199;Crooke, Biotechnology 1992, 10: 882-6). Apart from hybridizationstrategies, the application of ribozymes, i.e. RNA molecules that act asenzymes, destroying RNA that carries the message of disease has alsobeen described (see e.g. Barinaga, Science 1993, 262: 1512-1514). Inpreferred embodiments, the subject to be treated is a human, andtherapeutic antisense nucleic acids or derivatives thereof are directedagainst a human Ma onconeuronal antigen, particularly Ma2. It ispreferred that cells of the central nervous system, preferably thebrain, of a subject are treated in such a way. Cell penetration can beperformed by known strategies such as coupling of antisense nucleicacids and derivatives thereof to carrier particles, or the abovedescribed techniques. Strategies for administering targeted therapeuticoligodeoxynucleotides are known to those of skill in the art (see e.g.Wickstrom, Trends Biotechnol 1992, 10: 281-287). In some cases, deliverycan be performed by mere topical application. Further approaches aredirected to intracellular expression of antisense RNA. In this strategy,cells are transformed ex vivo with a recombinant gene that directs thesynthesis of an RNA that is complementary to a region of target nucleicacid. Therapeutical use of intracellularly expressed antisense RNA isprocedurally similar to gene therapy. A recently developed method ofregulating the intracellular expression of genes by the use ofdouble-stranded RNA, known variously as RNA interference (RNAi), can beanother effective approach for nucleic acid therapy (Hannon, Nature2002, 418: 244-251).

In further preferred embodiments, the method comprises grafting donorcells into the central nervous system, preferably the brain, of saidsubject, or donor cells preferably treated so as to minimize or reducegraft rejection, wherein said donor cells are genetically modified byinsertion of at least one transgene encoding said agent or agents. Saidtransgene might be carried by a viral vector, in particular a retroviralvector. The transgene can be inserted into the donor cells by a nonviralphysical transfection of DNA encoding a transgene, in particular bymicroinjection. Insertion of the transgene can also be performed byelectroporation, chemically mediated transfection, in particular calciumphosphate transfection or liposomal mediated transfection (see McCelland and Pardee, Expression Genetics: Accelerated and High-ThroughputMethods, Eaton Publishing, Natick, Mass., 1999).

In preferred embodiments, said agent for treating and preventing aneurodegenerative disease, in particular AD, is a therapeutic proteinwhich can be administered to said subject, preferably a human, by aprocess comprising introducing subject cells into said subject, saidsubject cells having been treated in vitro to insert a DNA segmentencoding said therapeutic protein, said subject cells expressing in vivoin said subject a therapeutically effective amount of said therapeuticprotein. Said DNA segment can be inserted into said cells in vitro by aviral vector, in particular a retroviral vector.

Methods of treatment, according to the present invention, comprise theapplication of therapeutic cloning, transplantation, and stem celltherapy using embryonic stem cells or embryonic germ cells, or neuronaladult stem cells, combined with any of the previously described cell-and gene therapeutic methods. Stem cells may be totipotent orpluripotent. They may also be organ-specific. Strategies for repairingdiseased and/or damaged brain cells or tissue comprise (i) taking donorcells from an adult tissue. Nuclei of those cells are transplanted intounfertilized egg cells from which the genetic material has been removed.Embryonic stem cells are isolated from the blastocyst stage of the cellswhich underwent somatic cell nuclear transfer. Use of differentiationfactors then leads to a directed development of the stem cells tospecialized cell types, preferably neuronal cells (Lanza et al., NatureMedicine 1999, 9: 975-977), or (ii) purifying adult stem cells, isolatedfrom the central nervous system, or from bone marrow (mesenchymal stemcells), for in vitro expansion and subsequent grafting andtransplantation, or (iii) directly inducing endogenous neural stem cellsto proliferate, migrate, and differentiate into functional neurons(Peterson DA, Curr. Opin. Pharmacol. 2002, 2: 34-42). Adult neural stemcells are of great potential for repairing damaged or diseased braintissues, as the germinal centers of the adult brain are free of neuronaldamage or dysfunction (Colman A, Drug Discovery World 2001, 7: 66-71).

In preferred embodiments, the subject for treatment or prevention,according to the present invention, can be a human, an experimentalanimal, e.g. a mouse or a rat, a domestic animal, or a non-humanprimate. The experimental animal can be an animal model for aneurodegenerative disorder, e.g. a transgenic mouse and/or a knock-outmouse with an Alzheimer's-type neuropathology.

In a further aspect, the invention features a modulator of an activity,or a level, or both said activity and said level of at least onesubstance which is selected from the group consisting of (i) a genecoding for a Ma onconeuronal antigen, and/or (ii) a transcriptionproduct of a gene coding for a Ma onconeuronal antigen and/or (iii) atranslation product of a gene coding for a Ma onconeuronal antigen,and/or (iv) a fragment, or derivative, or variant of (i) to (iii).

In an additional aspect, the invention features a pharmaceuticalcomposition comprising said modulator and preferably a pharmaceuticalcarrier. Said carrier refers to a diluent, adjuvant, excipient, orvehicle with which the modulator is administered.

In a further aspect, the invention features a modulator of an activity,or a level, or both said activity and said level of at least onesubstance which is selected from the group consisting of (i) a genecoding for a Ma onconeuronal antigen, and/or (ii) a transcriptionproduct of a gene coding for a Ma onconeuronal antigen, and/or (iii) atranslation product of a gene coding for a Ma onconeuronal antigen,and/or (iv) a fragment, or derivative, or variant of (i) to (iii) foruse in a pharmaceutical composition.

In another aspect, the invention provides for the use of a modulator ofan activity, or a level, or both said activity and said level of atleast one substance which is selected from the group consisting of (i) agene coding for a Ma onconeuronal antigen, and/or (ii) a transcriptionproduct of a gene coding for a Ma onconeuronal antigen and/or (iii) atranslation product of a gene coding for a Ma onconeuronal antigen,and/or (iv) a fragment, or derivative, or variant of (i) to (iii) for apreparation of a medicament for treating or preventing aneurodegenerative disease, in particular Alzheimer's disease.

In one aspect, the present invention also provides a kit comprising oneor more containers filled with a therapeutically or prophylacticallyeffective amount of said pharmaceutical composition.

In a further aspect, the invention features a recombinant, non-humananimal comprising a non-native gene sequence coding for a Maonconeuronal antigen, or a fragment thereof, or a derivative, or variantthereof. The generation of said recombinant, non-human animal comprises(i) providing a gene targeting construct containing said gene sequenceand a selectable marker sequence, and (ii) introducing said targetingconstruct into a stem cell of a non-human animal, and (iii) introducingsaid non-human animal stem cell into a non-human embryo, and (iv)transplanting said embryo into a pseudopregnant non-human animal, and(v) allowing said embryo to develop to term, and (vi) identifying agenetically altered non-human animal whose genome comprises amodification of said gene sequence in both alleles, and (vii) breedingthe genetically altered non-human animal of step (vi) to obtain agenetically altered non-human animal whose genome comprises amodification of said endogenous gene, wherein said gene ismis-expressed, or under-expressed, or over-expressed, and wherein saiddisruption or alteration results in said non-human animal exhibiting apredisposition to developing symptoms of neuropathology similar to aneurodegenerative disease, in particular Alzheimer's disease. Strategiesand techniques for the generation and construction of such an animal areknown to those of ordinary skill in the art (see e.g. Capecchi, Science1989, 244: 1288-1292 and Hogan et al., 1994, Manipulating the MouseEmbryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. and Jackson and Abbott, Mouse Genetics andTransgenics: A Practical Approach, Oxford University Press, Oxford,England, 1999). It is preferred to make use of such a recombinantnon-human animal as an animal model for investigating neurodegenerativediseases, in particular Alzheimer's disease. Such an animal may beuseful for screening, testing and validating compounds, agents andmodulators in the development of diagnostics and therapeutics to treatneurodegenerative diseases, in particular Alzheimer's disease.

In preferred embodiments, said recombinant, non-human animal comprises anon-native gene sequence coding for a member of the Ma onconeuronalantigen family, in particular Ma2, or a fragment, or derivative, orvariant thereof.

In another aspect, the invention features an assay for screening for amodulator of neurodegenerative diseases, in particular Alzheimer'sdisease, or related diseases and disorders of one or more substancesselected from the group consisting of (i) a gene coding for a Maonconeuronal antigen, and/or (ii) a transcription product of a genecoding for a Ma onconeuronal antigen, and/or (iii) a translation productof a gene coding for a Ma onconeuronal antigen, and/or (iv) a fragment,or derivative, or variant of (i) to (iii). This screening methodcomprises (a) contacting a cell with a test compound, and (b) measuringthe activity, or the level, or both the activity and the level of one ormore substances recited in (i) to (iv), and (c) measuring the activity,or the level, or both the activity and the level of said substances in acontrol cell not contacted with said test compound, and (d) comparingthe levels of the substance in the cells of step (b) and (c), wherein analteration in the activity and/or level of said substances in thecontacted cells indicates that the test compound is a modulator of saiddiseases and disorders.

In one further aspect, the invention features a screening assay for amodulator of neurodegenerative diseases, in particular Alzheimer'sdisease, or related diseases and disorders of one or more substancesselected from the group consisting of (i) a gene coding for a Maonconeuronal antigen, and/or (ii) a transcription product of a genecoding for a Ma onconeuronal antigen, and/or (iii) a translation productof a gene coding for a Ma onconeuronal antigen, and/or (iv) a fragment,or derivative, or variant of (i) to (iii), comprising (a) administeringa test compound to a test animal which is predisposed to developing orhas already developed symptoms of a neurodegenerative disease or relateddiseases or disorders, and (b) measuring the activity and/or level ofone or more substances recited in (i) to (iv), and (c) measuring theactivity and/or level of said substances in a matched control animalwhich is equally predisposed to developing or has already developed saidsymptoms and to which animal no such test compound has beenadministered, and (d) comparing the activity and/or level of thesubstance in the animals of step (b) and (c), wherein an alteration inthe activity and/or level of substances in the test animal indicatesthat the test compound is a modulator of said diseases and disorders.

In a preferred embodiment, said test animal and/or said control animalis a recombinant, non-human animal which expresses the gene coding for aMa onconeuronal antigen, or a fragment, or derivative, or variantthereof, under the control of a transcriptional regulatory element whichis not the native Ma onconeuronal antigen gene transcriptional controlregulatory element.

In another embodiment, the present invention provides a method forproducing a medicament comprising the steps of (i) identifying amodulator of neurodegenerative diseases by a method of theaforementioned screening assays and (ii) admixing the modulator with apharmaceutical carrier. However, said modulator may also be identifiableby other types of screening assays.

In another aspect, the present invention provides for an assay fortesting a compound, preferably for screening a plurality of compounds,for inhibition of binding between a ligand and a voltage-gated ionchannel, or a fragment, or derivative, or variant thereof. Saidscreening assay comprises the steps of (i) adding a liquid suspension ofsaid Ma onconeuronal antigen, or a fragment, or derivative, or variantthereof, to a plurality of containers, and (ii) adding a compound or aplurality of compounds to be screened for said inhibition to saidplurality of containers, and (iii) adding fluorescently labelled ligandto said containers, and (iv) incubating said Ma onconeuronal antigen, orsaid fragment, or derivative, or variant thereof, and said compound orplurality of compounds, and said fluorescently labelled ligand, and (v)measuring the amounts of fluorescence associated with said Maonconeuronal antigen, or with said fragment, or derivative, or variantthereof, and (vi) determining the degree of inhibition by one or more ofsaid compounds of binding of said ligand to said Ma onconeuronalantigen, or said fragment, or derivative, or variant thereof. Instead ofutilizing a fluorescently labelled ligand, it might in some aspects bepreferred to use any other detectable label known to the person skilledin the art, e.g. radioactive labels, and detect it accordingly. Saidmethod may be useful for the identification of novel compounds as wellas for evaluating compounds which have been improved or otherwiseoptimized in their ability to inhibit the binding of a ligand to a geneproduct of a gene coding for a Ma onconeuronal antigen, or a fragment,or derivative, or variant thereof. One example of a fluorescent bindingassay, in this case based on the use of carrier particles, is disclosedand described in patent application WO 00/52451. A further example isthe competitive assay method as described in patent WO 02/01226.Preferred signal detection methods for screening assays of the instantinvention are described in the following patent applications: WO96/13744, WO 98/16814, WO 98/23942, WO 99/17086, WO 99/34195, WO00/66985, WO 01/59436, WO 01/59416.

In one further embodiment, the present invention provides a method forproducing a medicament comprising the steps of (i) identifying acompound as an inhibitor of binding between a ligand and a gene productof a gene coding for a Ma onconeuronal antigen by the aforementionedinhibitory binding assay and (ii) admixing the compound with apharmaceutical carrier. However, said compound may also be identifiableby other types of screening assays.

In another aspect, the invention features an assay for testing acompound, preferably for screening a plurality of compounds to determinethe degree of binding of said compounds to a Ma onconeuronal antigen, orto a fragment, or derivative, or variant thereof. Said screening assaycomprises (I) adding a liquid suspension of said Ma onconeuronalantigen, or a fragment, or derivative, or variant thereof, to aplurality of containers, and (ii) adding a fluorescently labelledcompound or a plurality of fluorescently labelled compounds to bescreened for said binding to said plurality of containers, and (iii)incubating said Ma onconeuronal antigen, or said fragment, orderivative, or variant thereof, and said fluorescently labelled compoundor fluorescently labelled compounds, and (iv) measuring the amounts offluorescence associated with said Ma onconeuronal antigen, or with saidfragment, or derivative, or variant thereof, and (v) determining thedegree of binding by one or more of said compounds to said Maonconeuronal antigen, or said fragment, or derivative, or variantthereof. In this type of assay it might be preferred to use afluorescent label. However, any other type of detectable label mightalso be employed. Said method may be useful for the identification ofnovel compounds as well as for evaluating compounds which have beenimproved or otherwise optimized in their ability to bind to a Maonconeuronal antigen gene product or fragment, or derivative, or variantthereof.

In one further embodiment, the present invention provides a method forproducing a medicament comprising the steps of (i) identifying acompound as a binder to a gene product of a gene coding for a Maonconeuronal antigen by the aforementioned binding assays and (ii)admixing the compound with a pharmaceutical carrier. However, saidcompound may also be identifiable by other types of screening assays.

In another embodiment, the present invention provides for a medicamentobtainable by any of the methods according to the herein claimedscreening assays. In one further embodiment, the instant inventionprovides for a medicament obtained by any of the methods according tothe herein claimed screening assays.

The present invention features a protein molecule shown in SEQ ID NO: 3,or a fragment, or derivative, or variant thereof, for use as adiagnostic target for detecting a neurodegenerative disease, preferablyAlzheimer's disease.

The present invention further features a protein molecule shown in SEQID NO: 3, or a fragment, or derivative, or variant thereof, for use as ascreening target for reagents or compounds preventing, or treating, orameliorating a neurodegenerative disease, preferably Alzheimer'sdisease.

In all types of assays disclosed herein it is preferred to study amember of the Ma onconeuronal antigen gene family. It is particularlypreferred to conduct screening assays with the onconeuronal antigen Ma2.

The present invention features an antibody which is specificallyimmunoreactive with an immunogen, wherein said immunogen is atranslation product of the onconeuronal antigen Ma2 gene or a fragment,or derivative, or variant thereof. The immunogen may compriseimmunogenic or antigenic epitopes or portions of a translation productof said gene, wherein said immunogenic or antigenic portion of atranslation product is a polypeptide, and wherein said polypeptideelicits an antibody response in an animal, and wherein said polypeptideis immunospecifically bound by said antibody. Methods for generatingantibodies are well known in the art (see Harlow et al., Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1988). The term “antibody”, as employed in the presentinvention, encompasses all forms of antibodies known in the art, such aspolyclonal, monoclonal, chimeric, recombinatorial, anti-idiotypic,humanized, or single chain antibodies, as well as fragments thereof (seeDubel and Breitling, Recombinant Antibodies, Wiley-Liss, New York, N.Y.,1999). Antibodies of the present invention are useful, for instance, ina variety of diagnostic and therapeutic methods, based onstate-in-the-art techniques (see Harlow and Lane, Using Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1999 and Edwards R., Immunodiagnostics: A PracticalApproach, Oxford University Press, Oxford, England, 1999) such asenzyme-immuno assays (e.g. enzyme-linked immunosorbent assay, ELISA),radioimmuno assays, chemoluminescence-immuno assays, Western-blot,immunoprecipitation and antibody microarrays. These methods involve thedetection of translation products of the onconeuronal antigen Ma2 gene.

In a preferred embodiment of the present invention, said antibodies canbe used for detecting the pathological state of a cell in a sample froma subject, comprising immunocytochemical staining of said cell with saidantibody, wherein an altered degree of staining, or an altered stainingpattern in said cell compared to a cell representing a known healthstatus indicates a pathological state of said cell. Preferably, thepathological state relates to a neurodegenerative disease, in particularto Alzheimer's disease. Immuno-cytochemical staining of a cell can becarried out by a number of different experimental methods well known inthe art. It might be preferred, however, to apply an automated methodfor the detection of antibody binding, wherein the determination of thedegree of staining of a cell, or the determination of the cellular orsubcellular staining pattern of a cell, or the topological distributionof an antigen on the cell surface or among organelles and othersubcellular structures within the cell, are carried out according to themethod described in U.S. Pat. No. 6,150,173.

Other features and advantages of the invention will be apparent from thefollowing description of figures and examples which are illustrativeonly and not intended to limit the remainder of the disclosure in anyway.

FIG. 1 depicts the brain regions with selective vulnerability toneuronal loss and degeneration in Alzheimer's disease. Primarily,neurons within the inferior temporal lobe, the entorhinal cortex, thehippocampus, and the amygdala are subject to degenerative processes inAlzheimer's disease (Terry et al., Annals of Neurology 1981,10:184-192). These brain regions are mostly involved in the processingof learning and memory functions. In contrast, neurons within thefrontal cortex, the occipital cortex, and the cerebellum remain largelyintact and preserved from neurodegenerative processes in Alzheimer'sdisease. Brain tissues from the frontal cortex (F), the temporal cortex(T), and the hippocampus (H) of Alzheimer's disease patients andhealthy, age-matched control individuals were used for the hereindisclosed examples. For illustrative purposes, the image of a normalhealthy brain was taken from a publication by Strange (BrainBiochemistry and Brain Disorders, Oxford University Press, Oxford, 1992,p. 4).

FIG. 2 discloses the initial identification of the differentialexpression of the gene coding for Ma2 in a suppressive subtractivehybridization screen. The figure shows a clipping of a large-scale dotblot hybridization experiment. Individual cDNA clones from a temporallysubtracted library were arrayed onto a nylon membrane and hybridizedwith probes enriched for genes expressed in the frontal cortex (F) andthe temporal cortex (T) of an Alzheimer's disease patient. Ia) cloneT16-G10; Ib) clone T16-H01; IIa) clone T16-G02; IIb) clone T16-H02;IIIa) clone T16-G03; Ma2; IIIb) clone T16-H03. Note the significantlystronger intensity of the hybridization signal for Ma2 in panel (F) (seearrowhead) as compared to the signal in panel (T).

FIGS. 3 and 4 illustrate the verification of the differential expressionof the Ma2 gene in AD brain tissues by quantitative RT-PCR analysis.Quantification of RT-PCR products from RNA samples collected from thefrontal cortex (F) and the temporal cortex (T) of AD patients (FIG. 3 a)and samples from the frontal cortex (F) and the hippocampus (H) of ADpatients (FIG. 4 a) was performed by the LightCycler rapid thermalcycling technique. Likewise, samples of healthy, age-matched controlindividuals were compared (FIG. 3 b for frontal cortex and temporalcortex, FIG. 4 b for frontal cortex and hippocampus). The data werenormalized to the combined average values of a set of standard geneswhich showed no significant differences in their gene expression levels.Said set of standard genes consisted of genes for the ribosomal proteinS9, cyclophilin B, the transferrin receptor, GAPDH, and beta-actin. Thefigures depict the kinetics of amplification by plotting the cyclenumber against the amount of amplified material as measured by itsfluorescence. Note that the amplification kinetics of Ma2 cDNA fromboth, the frontal and temporal cortices of a normal control individual,and from the frontal cortex and hippocampus of a normal controlindividual, respectively, during the exponential phase of the reactionare juxtaposed (FIGS. 3 b and 4 b, arrowheads), whereas in Alzheimer'sdisease (FIGS. 3 a and 4 a, arrowheads) there is a significantseparation of the corresponding curves, indicating a differentialexpression of the Ma2 gene in the respective analyzed brain regions.

FIG. 5 depicts SEQ ID NO: 1, the nucleotide sequence of the 259 bp Ma2cDNA fragment, identified and obtained by suppressive subtractivehybridization cloning (sequence in 5′ to 3′ direction).

FIG. 6 charts the schematic alignment of SEQ ID NO: 1, the Ma2 cDNAfragment, to the nucleotide sequence of the Ma2 onconeuronal antigenKIAA0883 (GenBank accession number AB020690). The open rectanglerepresents the Ma2 open reading frame (ORF), thin bars represent the 5′and 3′ untranslated regions (UTR), respectively. The Ma2 cDNA fragmentis located within the 3′ UTR of the mRNA.

FIG. 7 renders the sequence alignment of SEQ ID NO: 1, the 259 bp Ma2cDNA fragment, with the nucleotide sequence of the Ma2 onconeuronalantigen, SEQ ID NO: 2, KIM0883 (GenBank accession number AB020690).

FIG. 8 shows SEQ ID NO: 2, the nucleotide sequence of the Ma2 cDNA,comprising 4253 nucleotides, GenBank accession number AB020690.

FIG. 9 discloses SEQ ID NO: 3, the amino acid sequence of Ma2 protein(GenBank accession number 094959, KIAA0883 protein). The full-length Ma2protein comprises 364 amino acids.

FIG. 10 depicts human cerebral cortex labeled with an affinity-purifiedrabbit anti-Ma2 antiserum (green signals) raised against a peptidecorresponding to amino acids 275 to 290 of the Ma2 protein.Immunoreactivity of Ma2 was detected in the pre-central cortex (CT) andin the white matter (WM) (FIG. 10 a, low magnification). The cortexshowed intense staining of neuronal cell processes and weak staining ofthe cytoplasma of some neurons (FIG. 10 b, high magnification). The sameimmunostaining pattern was also obtained by using another antiserumraised against a peptide mapping to amino acids 304 to 317 of the Ma2protein. Blue signals indicate nuclei stained with DAPI.

Table 1 lists the gene expression levels in the frontal cortex relativeto the temporal cortex for the Ma2 gene in seven Alzheimer's diseasepatients, herein identified by internal reference numbers P010, P011,P012, P014, P016, P017, P019 (1.62 to 5.86 fold) and five healthy,age-matched control individuals, herein identified by internal referencenumbers C005, C008, C011, C012, C014 (0.66 to 1.29 fold). The valuesshown are reciprocal values according to the formula described herein.

Table 2 lists the Ma2 gene expression levels in the frontal cortexrelative to the hippocampus in six Alzheimer's disease patients, hereinidentified by internal reference numbers P010, P011, P012, P014, P016,P019 (0.93 to 2.50 fold) and three healthy, age-matched controlindividuals, herein identified by internal reference numbers C004, C005,C008 (0.92 to 1.02 fold). The values shown are reciprocal valuesaccording to the formula described herein (see below).

EXAMPLE 1

(i) Brain Tissue Dissection from Patients with Alzheimer's Disease:

Brain tissues from Alzheimer's disease patients and age-matched controlsubjects were collected within 6 hours post-mortem and immediatelyfrozen on dry ice. Sample sections from each tissue were fixed inparaformaldehyde for histopathological confirmation of the diagnosis.Brain areas for differential expression analysis were identified (seeFIG. 1) and stored at −80° C. until RNA extractions were performed.

(ii) Isolation of Total RNA:

Total RNA was extracted from post-mortem brain tissue by using theRNeasy kit (Qiagen) according to the manufacturer's protocol. Theaccurate RNA concentration and the RNA quality were determined with theDNA LabChip system using the Agilent 2100 Bioanalyzer (AgilentTechnologies).

For additional quality testing of the prepared RNA, i.e. exclusion ofpartial degradation and testing for DNA contamination, specificallydesigned intronic GAPDH oligonucleotides and genomic DNA as referencecontrol were used to generate a melting curve with the LightCyclertechnology as described in the corresponding protocol (Roche).

(iii) cDNA Synthesis and Identification of Differentially ExpressedGenes by Suppressive Subtractive Hybridization:

This technique compares two populations of mRNA and provides clones ofgenes that are expressed in one population but not in the other. Theapplied technique was described in detail by Diatchenko et al. (Proc.Natl. Acad. Sci. USA 1996, 93: 6025-30). In the present invention, mRNApopulations from post-mortem brain tissues from Alzheimer's diseasepatients were compared. Specifically, mRNA of the frontal cortex wassubtracted from mRNA of the inferior temporal cortex. The necessaryreagents were taken from the PCR-Select cDNA subtraction kit (Clontech),and all steps were performed as described in the manufacturer'sprotocol. Specifically, 2 μg mRNA each were used for first-strand andsecond-strand cDNA synthesis. After Rsal-digestion and adaptor ligationhybridization of tester and driver was performed for 8 hours (firsthybridization) and 15 hours (second hybridization) at 68° C. Two PCRsteps were performed to amplify differentially expressed genes (firstPCR: 27 cycles of 94° C. and 30 sec, 66° C. and 30 sec, and 72° C. and1.5 min; nested PCR: 12 cycles of 94° C. and 30 sec, 66° C. and 30 sec,and 72° C. and 1.5 min) using adaptor specific primers (included in thesubtraction kit) and 50×Advantage Polymerase Mix (Clontech).Efficiencies of Rsal-digestions, adaptor ligations and subtractivehybridizations were checked as recommended in the kit. Subtracted cDNAswere inserted into the pCR vector and transformed into E. coli INVαF′cells (Invitrogen).

To isolate individual cDNAs of the subtracted library, single bacterialtransformants were incubated in 100 μl LB (with 50 μg/ml ampicillin) at37° C. for at least 4 hours. Inserts were PCR amplified (95° C. and 30sec, 68° C. and 3 min for 30 cycles) in a volume of 20 μl containing 10mM Tris-HCl pH 9.0, 1.5 mM MgCl₂, 50 mM KCl, 200 μM dNTP, 0.5 μM adaptorspecific primers (included in the subtraction kit), 1.5 Units Taqpolymerase (Pharmacia Biotech), and 1 μl of bacterial culture.

1.5 μl of a mixture containing 3 μl PCR amplified inserts and 2 μl 0.3 NNaOH/15% Ficoll were spotted onto a positively charged nylon membrane(Roche). In this way, hundreds of spots were arrayed on duplicatefilters for subsequent hybridization analysis. The differentialscreening step consisted of hybridizations of the subtracted librarywith itself to minimize background (Wang and Brown, Proc. Natl. Acad.Sci. USA 1991, 88: 11505-9). The probes were generated from the nestedPCR product of the subtraction following the instructions of theClontech subtraction kit. Labeling with digoxigenin was performed withthe DIG DNA Labeling Kit (Roche). Hybridizations were carried outovernight in DIG Easy HYB (Roche) at 43° C. The filters were washedtwice in 2×SSC/0.5% SDS at 68° C. for 15 min and twice in 0.1×SSC/0.5%SDS at 68° C. for 15 min, and subjected to detection using anti-DIG-APconjugates and CDP-Star as chemiluminescent substrate according to theinstructions of the DIG DNA Detection Kit (Roche). Blots were exposed toKodak Biomax MR chemiluminescent film at room temperature for severalminutes. The nucleotide sequences of clones of interest were obtainedusing methods well known to those skilled in the art. For nucleotidesequence analyses and homology searches, computer algorithms of theUniversity of Wisconsin Genetics Computer Group (GCG) in conjunctionwith publicly available nucleotide and peptide sequence information(Genbank and EMBL databases) were employed. The results of one suchsubtractive hybridization experiment for the Ma2 gene are shown in FIG.2.

(iv) Confirmation of Differential Expression by Quantitative RT-PCR:

Positive corroboration of differential expression of the Ma2 gene wasperformed using the LightCycler technology (Roche). This techniquefeatures rapid thermal cyling for the polymerase chain reaction as wellas real-time measurement of fluorescent signals during amplification andtherefore allows for highly accurate quantification of RT-PCR productsby using a kinetic, rather than an endpoint readout. The ratios of Ma2cDNA from the temporal cortex and frontal cortex, and from thehippocampus and frontal cortex, respectively, were determined (relativequantification).

First, a standard curve was generated to determine the efficiency of thePCR with specific primers for Ma2: 5′-GTTGCATGACATCTGGAACACA-3′ and5′-GAGCAGACAGGAACATCGTGAA-3′.

PCR amplification (95° C. and 1 sec, 56° C. and 5 sec, and 72° C. and 5sec) was performed in a volume of 20 μl containing Lightcycler-FastStartDNA Master SYBR Green I ready-to-use mix (contains FastStart Taq DNApolymerase, reaction buffer, dNTP mix with dUTP instead of dTTP, SYBRGreen I dye, and 1 mM MgCl₂, Roche), 0.5 μM primers, 2 μl of a cDNAdilution series (final concentration of 40, 20, 10, 5, 1, and 0.5 nghuman total brain cDNA, Clontech) and, depending on the primers used,additional 3 mM MgCl₂. Melting curve analysis revealed a single peak atapproximately 80.5° C. with no visible primer dimers. Quality and sizeof the PCR product were determined with the DNA LabChip system (Agilent2100 Bioanalyzer, Agilent Technologies). A single peak at the expectedsize of 77 bp was observed in the electropherogram of the sample.

In an analogous manner, the PCR protocol was applied to determine thePCR efficiency of a set of reference genes which were selected as areference standard for quantification. In the present invention, themean value of five such reference genes was determined: (1) cyclophilinB, using the specific primers 5′-ACTGAAGCACTACGGGCCTG-3′ and5′-AGCCGTTGGTGTCTTTGCC-3′ except for MgCl₂ (an additional 1 mM was addedinstead of 3 mM). Melting curve analysis revealed a single peak atapproximately 87° C. with no visible primer dimers. Agarose gel analysisof the PCR product showed one single band of the expected size (62 bp).(2) Ribosomal protein S9 (RPS9), using the specific primers5′-GGTCAAATTTACCCTGGCCA-3′ and 5′-TCTCATCAAGCGTCAGCAGTTC-3′ (exception:additional 1 mM MgCl₂ was added instead of 3 mM). Melting curve analysisrevealed a single peak at approximately 85° C. with no visible primerdimers. Agarose gel analysis of the PCR product showed one single bandwith the expected size (62 bp). (3) beta-actin, using the specificprimers 5′-TGGAACGGTGAAGGTGACA-3′ and 5′-GGCAAGGGACTTCCTGTAA-3′. Meltingcurve analysis revealed a single peak at approximately 87° C. with novisible primer dimers. Agarose gel analysis of the PCR product showedone single band with the expected size (142 bp). (4) GAPDH, using thespecific primers 5′-CGTCATGGGTGTGMCCATG-3′ and5′-GCTAAGCAGTTGGTGGTGCAG-3′. Melting curve analysis revealed a singlepeak at approximately 83° C. with no visible primer dimers. Agarose gelanalysis of the PCR product showed one single band with the expectedsize (81 bp). (5) Transferrin receptor TRR, using the specific primers5′-GTCGCTGGTCAGTTCGTGATT-3′ and 5′-AGCAGTTGGCTGTTGTACCTCTC-3′. Meltingcurve analysis revealed a single peak at approximately 83° C. with novisible primer dimers. Agarose gel analysis of the PCR product showedone single band with the expected size (80 bp).

For calculation of the values, first the logarithm of the cDNAconcentration was plotted against the threshold cycle number C_(t) forthe Ma2 gene and the five reference standard genes. The slopes and theintercepts of the standard curves (i.e. linear regressions) werecalculated for all genes. In a second step, cDNA from temporal cortexand frontal cortex, and from hippocampus and frontal cortex,respectively, were analyzed in parallel and normalized to cyclophilin B.The C_(t) values were measured and converted to ng total brain cDNAusing the corresponding standard curves:10{circumflex over ( )}((C _(t) value−intercept)/slope)[ng total braincDNA]

The values of temporal and frontal cortex Ma2 cDNAs, and the values ofhippocampus and frontal cortex Ma2 cDNAs, respectively, were normalizedto cyclophilin B, and the ratios were calculated according to formulas:$\begin{matrix}{{Ratio} = \frac{{Ma2}\quad{{{temporal}\quad\lbrack{ng}\rbrack}/{cyclophilin}}\quad B\quad{{temporal}\quad\lbrack{ng}\rbrack}}{{Ma2}\quad{{{frontal}\quad\lbrack{ng}\rbrack}/{cyclophilin}}\quad B\quad{{frontal}\quad\lbrack{ng}\rbrack}}} \\{{Ratio} = \frac{{Ma2}\quad{{{hippocampus}\quad\lbrack{ng}\rbrack}/{cyclophilin}}\quad B\quad{{hippocampus}\quad\lbrack{ng}\rbrack}}{{Ma2}\quad{{{frontal}\quad\lbrack{ng}\rbrack}/{cyclophilin}}\quad B\quad{{frontal}\quad\lbrack{ng}\rbrack}}}\end{matrix}$

In a third step, the set of reference standard genes was analyzed inparallel to determine the mean average value of the temporal to frontalratios, and of the hippocampal to frontal ratios, respectively, ofexpression levels of the reference standard genes for each individualbrain sample. As cyclophilin B was analyzed in step 2 and step 3, andthe ratio from one gene to another gene remained constant in differentruns, it was possible to normalize the values for Ma2 to the meanaverage value of the set of reference standard genes instead ofnormalizing to one single gene alone. The calculation was performed bydividing the respective ratio shown above by the deviation ofcyclophilin B from the mean value of all housekeeping genes. The resultsof such quantitative RT-PCR analysis for the Ma2 gene are shown in FIGS.3 and 4.

(v) Immunohistochemistry:

For immunofluorescence staining of Ma2 in human brain, frozen sectionswere prepared from post-mortem pre-central gyrus of a donor person(Cryostat Leica CM3050S) and fixed in 4% Paraformaldehyd (PFA) for 20min. After washing in PBS, the sections were pre-incubated with blockingbuffer (10% normal goat serum, 0.2% Triton X-100 in PBS) for 30 min, andthen incubated with affinity-purified rabbit anti-Ma2 antisera(1:20-1:40 diluted in blocking buffer, custom-made by DavidsBiotechnologies, Regensburg) overnight at 4° C. After rinsing threetimes in 0.1% Triton X-100/PBS, the sections were incubated withFITC-conjugated goat anti-rabbit IgG (1:150 diluted in 1% BSA/PBS) for 2hours at room temperature, and then again washed in PBS. Staining of thenuclei was performed by incubation of the sections with 51M DAPI in PBSfor 3 min (blue signal). In order to block the autofluoresence oflipofuscin in human brain, the sections were treated with 1% Sudan BlackB in 70% ethanol for 2-10 min at room temperature, sequentially dippedin 70% ethanol, destined water and PBS. The sections were coverslippedby ‘Vectrashield mounting medium’ (Vector Laboratories, Burlingame,Calif.) and observed under an inverted microscope (IX81, OlympusOptical). The digital images were captured with the appropriate software(AnalySiS, Olympus Optical).

1. A method of diagnosing or prognostication a neurodegenerative diseasein a subject, or determining whether a subject is at increased risk ofdeveloping said disease, comprising: determining a level and/or anactivity of (i) a transcription product of a gene coding for a Maonconeuronal antigen, and/or (ii) a translation product of a gene codingfor a Ma onconeuronal antigen and/or (iii) a fragment, or derivative, orvariant of said transcription or translation product, in a sample fromsaid subject and comparing said level and/or said activity to areference value representing a known disease or health status, therebydiagnosing or prognosticating said neurodegenerative disease in saidsubject, or determining whether said subject is at increased risk ofdeveloping said neurodegenerative disease.
 2. A method of monitoring theprogression of a neurodegenerative disease in a subject, comprising:determining a level and/or an activity of (i) a transcription product ofa gene coding for a Ma onconeuronal antigen, and/or (ii) a translationproduct of a gene coding for a Ma onconeuronal antigen, and/or (iii) afragment, or derivative, or variant of said transcription or translationproduct, in a sample from said subject and comparing said level and/orsaid activity to a reference value representing a known disease orhealth status, thereby monitoring the progression of saidneurodegenerative disease in said subject.
 3. A method of evaluating atreatment for a neurodegenerative disease, comprising: determining alevel and/or an activity of (i) a transcription product of a gene codingfor a Ma onconeuronal antigen, and/or (ii) a translation product of agene coding for a Ma onconeuronal antigen, and/or (iv) a fragment, orderivative, or variant of said transcription or translation product, ina sample from a subject being treated for said disease and comparingsaid level and/or said activity to a reference value representing aknown disease or health status, thereby evaluating said treatment forsaid neurodegenerative disease.
 4. The method according to claim 1wherein said neurodegenerative disease is Alzheimer's disease.
 5. Themethod according to claim 1 wherein said Ma onconeuronal antigen is theMa2 onconeuronal antigen.
 6. The method according to claim 1 whereinsaid sample comprises a cell, or a tissue, or an organ, or a body fluid,in particular cerebrospinal fluid or blood.
 7. The method according toclaim 1 wherein said reference value is that of a level and/or anactivity of (i) a transcription product of a gene coding for a Maonconeuronal antigen, and/or (ii) a translation product of a gene codingfor a Ma onconeuronal antigen, and/or (iii) a fragment, or derivative,or variant of said transcription or translation product, in a samplefrom a subject not suffering from said neurodegenerative disease.
 8. Themethod according to claim 1 wherein an alteration in Ma onconeuronalantigen mRNA and/or protein in a cell, or tissue, or body fluid fromsaid subject relative to a reference value representing a known healthstatus indicates a diagnosis, or prognosis, or increased risk ofAlzheimer's disease in said subject.
 9. A kit for diagnosing orprognosticating a neurodegenerative disease, in particular Alzheimer'sdisease, in a subject, or determining the propensity or predispositionof a subject to develop such a disease, said kit comprising: (a) atleast one reagent which is selected from the group consisting of (i)reagents that selectively detect a transcription product of a genecoding for a Ma onconeuronal antigen (ii) reagents that selectivelydetect a translation product of a gene coding for a Ma onconeuronalantigen and (b) instruction for diagnosing, or prognosticating aneurodegenerative disease, in particular Alzheimer's disease, ordetermining the propensity or predisposition of a subject to developsuch a disease by detecting a level, or an activity, or both said leveland said activity, of said transcription product and/or said translationproduct of a gene coding for a Ma onconeuronal antigen, in a sample fromsaid subject; and diagnosing or prognosticating a neurodegenerativedisease, in particular Alzheimer's disease, or determining thepropensity or predisposition of said subject to develop such a disease,wherein a varied level, or activity, or both said level and saidactivity, of said transcription product and/or said translation productcompared to a reference value representing a known health status; or alevel, or activity, or both said level and said activity, of saidtranscription product and/or said translation product similar or equalto a reference value representing a known disease status indicates adiagnosis or prognosis of a neurodegenerative disease, in particularAlzheimer's disease, or an increased propensity or predisposition ofdeveloping such a disease.
 10. A method of treating or preventing aneurodegenerative disease, in particular Alzheimer's disease, in asubject comprising administering to said subject in a therapeutically orprophylactically effective amount an agent or agents which directly orindirectly affect an activity and/or a level of (i) a gene coding for aMa onconeuronal antigen, and/or (ii) a transcription product of a genecoding for a Ma onconeuronal antigen, and/or (iii) a translation productof a gene coding for a Ma onconeuronal antigen, and/or (iv) a fragment,or derivative, or variant of (i) to (iii).
 11. A modulator of anactivity and/or of a level of at least one substance which is selectedfrom the group consisting of (i) a gene coding for a Ma onconeuronalantigen and/or (ii) a transcription product of a gene coding for a Maonconeuronal antigen and/or (iii) a translation product of a gene codingfor a Ma onconeuronal antigen, and/or (iv) a fragment, or derivative, orvariant of (i) to (iii).
 12. Use of a modulator of an activity and/or ofa level of at least one substance which is selected from the groupconsisting of (i) a gene coding for a Ma onconeuronal antigen, and/or(ii) a transcription product of a gene coding for a Ma onconeuronalantigen, and/or (iii) a translation product of a gene coding for a Maonconeuronal antigen, and/or (iv) a fragment, or derivative, or variantof (i) to (iii) for a preparation of a medicament for treating orpreventing a neurodegenerative disease, in particular Alzheimer'sdisease.
 13. A recombinant, non-human animal comprising a non-nativegene sequence coding for a Ma onconeuronal antigen or a fragmentthereof, or a derivative, or a variant thereof, said animal beingobtainable by: (i) providing a gene targeting construct comprising saidgene sequence and a selectable marker sequence, and (ii) introducingsaid targeting construct into a stem cell of a non-human animal, and(iii) introducing said non-human animal stem cell into a non-humanembryo, and (iv) transplanting said embryo into a pseudopregnantnon-human animal, and (v) allowing said embryo to develop to term, and(vi) identifying a genetically altered non-human animal whose genomecomprises a modification of said gene sequence in both alleles, and(vii) breeding the genetically altered non-human animal of step (vi) toobtain a genetically altered non-human animal whose genome comprises amodification of said endogenous gene, wherein said disruption results insaid non-human animal exhibiting a predisposition to developing symptomsof a neurodegenerative disease or related diseases or disorders.
 14. Theanimal according to claim 13 wherein said Ma onconeuronal antigen isMa2.
 15. Use of the recombinant, non-human animal according to claim 13and 14 for screening, testing, and validating compounds, agents, andmodulators in the development of diagnostics and therapeutics to treatneurodegenerative diseases, in particular Alzheimer's disease.
 16. Anassay for screening for a modulator of neurodegenerative diseases, inparticular Alzheimer's disease, or related diseases or disorders of oneor more substances selected from the group consisting of (i) a genecoding for a Ma onconeuronal antigen, and/or (ii) a transcriptionproduct of a gene coding for a Ma onconeuronal antigen, and/or (iii) atranslation product of a gene coding for a Ma onconeuronal antigen,and/or (iv) a fragment, or derivative, or variant of (i) to (iii), saidmethod comprising: (a) contacting a cell with a test compound; (b)measuring the activity and/or level of one or more substances recited in(i) to (iv); (c) measuring the activity and/or level of one or moresubstances recited in (i) to (iv) in a control cell not contacted withsaid test compound; and (d) comparing the levels and/or activities ofthe substance in the cells of step (b) and (c), wherein an alteration inthe activity and/or level of substances in the contacted cells indicatesthat the test compound is a modulator of said diseases or disorders. 17.A protein molecule, said protein molecule being a translation product ofthe gene coding for a Ma onconeuronal antigen, or a fragment, orderivative, or variant thereof, for use as a diagnostic target fordetecting a neurodegenerative disease, preferably Alzheimer's disease.18. A protein molecule, said protein molecule being a translationproduct of the gene coding for a Ma onconeuronal antigen, or a fragment,or derivative, or variant thereof, for use as a screening target forreagents or compounds preventing, or treating, or ameliorating aneurodegenerative disease, preferably Alzheimer's disease.
 19. Use of anantibody specifically immunoreactive with an immunogen, wherein saidimmunogen is a translation product of the Ma2 gene, or a fragment, or aderivative, or variant thereof, for detecting the pathological state ofa cell in a sample from a subject, comprising immunocytochemicalstaining of said cell with said antibody, wherein an altered degree ofstaining, or an altered staining pattern in said cell compared to a cellrepresenting a known health status indicates a pathological state ofsaid cell, and wherein said pathological state relates to aneurodegenerative disease, preferably Alzheimer's disease.